PROJECT SUMMARY Signals present within the tumor microenvironment (TME) undermine the ability of the immune system to fight cancer. In particular, these signals together with chronic stimulation disable effector CD8+ T cell responses by promoting the development of a dysfunctional or ?exhausted? T cell state. As dysfunctional CD8+ T cells are defective in their ability to elicit cytotoxicity and produce pro-inflammatory cytokines, they are poor mediators of tumor clearance. Moreover, dysfunctional CD8+ T cells can produce IL-10, indicating that they can also contribute to immune suppression with the TME. Thus, dysfunctional CD8+ T cells present not only an obstacle but also a liability for the generation of productive anti-tumor immunity. Accordingly, understanding both the T cell intrinsic and extrinsic signals that promote dysfunctional phenotype is of key importance in devising novel therapies to improve anti-cancer T cell responses. We have examined the gene programs underlying T cell dysfunction in CD8+ tumor-infiltrating lymphocytes (TILs). We have thus discovered that NR3C1, the gene encoding the glucocorticoid receptor (GR), is preferentially expressed by CD8+ TILs that exhibit severe dysfunctional phenotype. Glucocorticoids (GCs) are steroid hormones and ligands for GR. Although both natural and synthetic glucocorticoids are known to be potent immune-suppressive agents, the precise molecular mechanisms by which they suppress effector T cell responses are poorly understood. Our preliminary data indicate that GC-GR signaling is indeed active in CD8+ TILs and that tumor-associated macrophages (TAMs) are a local source of steroid in the TME. We further find that mice that lack expression of NR3C1 specifically in CD8+ T cells exhibit improved tumor growth control and that NR3C1-deficient CD8+ TILs exhibit improved effector function concomitant with dramatically reduced expression of co-inhibitory receptors such as Tim-3 and PD-1. In line with these data, we find that GC-GR signaling promotes co-inhibitory receptor expression and dampens both the cytotoxic capacity and pro-inflammatory cytokine production of CD8+ T cells in vitro. Interestingly, we find that GC-GR signaling potently synergizes with the immunoregulatory cytokine IL-27 to further augment the expression of co-inhibitory receptors and dampen pro-inflammatory cytokine production and cytotoxic capacity in CD8+ T cells while concomitantly inducing IL-10 production. Based on our preliminary data, we hypothesize that GC-GR signaling is a key component of the immune suppressive network in the TME that disables anti-tumor T cell responses by: 1) direct promotion of dysfunction-associated gene programs in effector T cells; and 2) synergizing with signals in the TME such as IL-27. We propose the following specific aims: 1) Molecularly dissect the role of steroid signaling in CD8+ and CD4+ T cells in the TME and 2) Define the GC-GR signaling circuit and the basis for synergy with IL-27 in immune- suppression in the TME.